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| − | {{USP-Brazil}} | + | {{USP-Brazil/CSS2}} |
| | + | {{USP-Brazil/PROJECT}} |
| | <html> | | <html> |
| − | | + | <div class="container conteudo"> |
| − | | + | <div id="menu-lateral-container"> |
| − | <div class="column full_size judges-will-not-evaluate"> | + | <div id="ancoramento"></div> |
| − | <h3>★ ALERT! </h3> | + | <sidenav id="menu-lateral"> |
| − | <p> This page is used by the judges to evaluate your team for the <a href=" https:// 2018.igem.org/ Judging/ Medals"> medal criterion</a> or <a href=" https:// 2018.igem.org/ Judging/ Awards"> award listed below</a>. </p> | + | |
| − | <p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p> | + | <ul class="barra-lateral"><h4>Project</h4> |
| − | </div> | + | <li><a href="#overview">Overview</a></li> |
| − | | + | <li><a href="#apply">Applications</a></li> |
| − | | + | <li><a href="#design">Project Design</a></li> |
| − | <div class="clear"></div> | + | <li><a href="#parts">Parts</a></li> |
| − | | + | </ul> |
| − | | + | </sidenav> |
| − | <div class="column full_size"> | + | </div> |
| − | <h1>Improve</h1>
| + | <div class="linha"> |
| − | <p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p> | + | <div class="coluna col12 cont-inter"> |
| − | | + | <main class="project"> |
| − | <h3>Gold Medal Criterion #2</h3>
| + | <article> |
| − | <p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry. | + | <div class="project-content applications"> |
| − | | + | <h2><a name="apply">Part Improvement</a></h2> |
| − | The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
| + | <p> We improved the part <a href=" http:// parts.igem.org/ wiki/ index.php?title=Part:BBa_E0022"> BBa_E0430</a> , an EYFP output device, by joining it with a constitutively expressed (by ptet, <a href=" http:// parts.igem.org/ wiki/ index.php?title=Part:BBa_R0040"> BBa_R0040</a> ) ECFP gene, to be used as a control for normalization of the output measurement, as this control gene would be subject to the same influences as the reporter gene (eg. cell density, plasmid copy number, metabolic load, environment properties). Instead of dividing the YFP measurement by the OD, as usual, dividing the construct's YFP output by the CFP output gives us better precision and an adimensional value for promoter strength. This method was used following the efforts of Rudge et al. on characterizing intrinsic promoter properties. |
| − | | + | </p> |
| − | <br><br> | + | <div class="column half_size"> |
| − | <b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p> | + | <p> This fluorescent protein pair is good for this type of measurement due to their spectral separation. In our experiments, we used excitation/emission values of 430/480 for CFP and 500/ 530 for YFP. The CFP protein has an LVA degradation tag, which should make the CFP measure more accurate as a representation of the immediate state of the gene's activity, independing of non-degraded, previous values. |
| − | | + | </p> |
| − | | + | </div> |
| − | </div> | + | <div class="column half_size"> |
| − | | + | <center><img src="https://static.igem.org/mediawiki/2018/3/38/T--USP-Brazil--Dual_Fluo_Spectrum.png" style="width:95%"></center> |
| − | | + | </div> |
| − | | + | <div class=" column full_size"> |
| | + | <center><img src="https://static.igem.org/mediawiki/2018/0/06/T--USP-Brazil--cfp.png.png"></center> |
| | + | <p> Unfortunately, this makes the CFP overall fluorescence lower and creates the need for a more precise measurement, rather than just measuring from LB. We have obtained good results while using LB and centrifuging the samples then ressuspending them in H2O, or while growing the cells in M9 medium. |
| | + | </p> |
| | + | <p> Our new part is < a href="http:/ /parts. igem. org/wiki/index.php?title=Part :BBa_K2771020">BBa_K2771020</a>, which still preserves de modularity property of the original part, as other teams can insert promoter parts upstream of our part and readily have a good reporter gene for the activity of one promoter. As we showed in our <a href="https://2018.igem.org/Team:USP-Brazil/Measurement">Measurement</a> page, this method also allows for a more constant value for controls and constitutive expression, having a smaller standard deviation relative to the mean, when compared to the same data when using OD as a normalization factor. We also built parts using quorum sensing promoters and characterized their activity using this improvement. |
| | + | </p> |
| | + | <center><img src="https://static.igem.org/mediawiki/2018/b/be/T--USP-Brazil--measurement_abstract.png" style="width:80%"></center> |
| | + | <h3>References</h3> |
| | + | <ul> |
| | + | <li>T.J.Rudge, J.R.Brown, F.Federici, N.Dalchau, A.Phillips, J.W.Ajioka, J.Haseloff. “Characterization of Intrinsic Properties of Promoters” ACS Synthetic Biology 5 (1), 89-98, (2016) doi:10.1021/acssynbio.5b00116 </li> |
| | + | </ul> |
| | + | </div> |
| | + | </div> |
| | + | </article> |
| | + | </main> |
| | + | </div> |
| | + | </div> |
| | + | </div> |
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| | </html> | | </html> |
| | + | {{USP-Brazil/FOOTER}} |
